Project Summary As a result of technical advances in the last decade, single-particle cryo-electron microscopy (cryo-EM) has emerged as a powerful approach to obtaining near atomic resolution structures of large biomolecular complexes, membrane proteins, and other biologically and biomedically important targets that in many cases have been intractable to X-ray crystallography. Cryo-EM requires only small amounts of biomolecule dispersed in solution and has become a first choice for initial attempts at structure determination. Enormous investments have been and continue to be made in new microscopes and cryo-EM facilities at universities and biomedical research institutes in the US and around the world. However, current sample preparation and handling protocols ? largely based on work from the 1980s, are complex, time consuming, error-prone, and often yield suboptimal outcomes. They are a major factor limiting efficient use of these expensive new facilities. This Phase I SBIR project builds on insights into fundamental processes of cryocooling, vitrification, and the thermomechanical response of multicomponent systems, and on MiTeGen's extensive expertise in development of tools and instruments for microsample manipulation and cryocooling. New sample holding supports will be developed that increase maximum sample cooling rates and sample quality, reduce mechanical stresses during cooling that are the primary source of resolution-degrading sample motion in the electron beam, facilitate characterization and optimization of sample preparation and cryocooling processes, and enable sample identification and tracking. A new automated sample cooling instrument will be developed that uses liquid nitrogen rather than liquid ethane as the primary sample coolant while delivering outstanding sample vitrification performance. The longer-term goal of this project is to deliver an integrated cryo-EM solution comprised of improved sample supports and tools for their safe handling, storage, and tracking, improved tools for sample thinning/removal, and an automated sample cryocooling and cryostorage system with integrated sample tracking that eliminates manual manipulations, provides a stable, contaminant-free cold chain from sample plunging to the microscope, and that substantially improves the efficiency of cryo-EM structure determination pipelines.